Congenital heart disease is not only surprisingly prevalent (~1% of live births), but is deadly (most common congenital cause of death). While mechanical and bioprosthetic valves work relatively well in adult patients, mechanical valves require anticoagulation not compatible with a child's activities and bioprosthetic valves quickly calcify in children. Even if these complications could be resolved, neither of these valve replacements can grow with the child. Therefore, a child must undergo open-heart surgery every several years to replace the valve that has been "outgrown." A tissue engineered heart valve (TEHV) would dramatically improve the lives of these patients. If cells from a given patient could be grown on a scaffold with the right signals, an implantable valve could be formed that could grow with the patient, making such repeated open-heart surgeries obsolete. While age-specific TEHVs are clearly needed, their complex structure, necessary for proper function, and how this structure changes with age has not been well characterized. In the first aim of this proposal layer, region, valve, and age-specific characterization of different matrix components will be performed on MV and AV from pigs ranging from the equivalent of young to elderly humans. Mechanical characterization will also be performed using uniaxial tensile testing and AFM compressive indentation. One of the largest challenges involved in making a TEHV is controlling cell phenotype, which can be done in a number of ways including by modifying the mechanical properties of a cell's substrate. Therefore in the second aim of this proposal, cells of each valve region and a range of ages will be seeded on hydrogel scaffolds of varying stiffnesses to determine the "optimal" stiffness for each. Then the ability to use stiffness to modify cell phenotype will be examined by seeding "young" valve cells of a given region on hydrogels with an "elderly" stiffness, and by seeding cells of a given region on a stiffness correlating to a different region. Relevance to Public Health: This research proposal addresses the considerable burden of congenital valve disease, a prevalent disease with suboptimal treatment options. These studies will provide a comprehensive view of the elements needed for a successful age-specific TEHV. Consideration will be paid not only to biological factors, but mechanical properties and how these characteristics can be manipulated in the context of the complex, heterogeneous and changing valve. Such studies are crucial before a successful TEHV can be created. The goals of this proposal are clearly in line with those of the NHLBI to understand the causes and develop interventions for debilitating cardiac diseases.